About 75% of birth defects involve the head, face, and oral tissues. Although orofacial clefts and other craniofacial malformations have clear environmental and genetic causes, insufficient information exists concerning the mechanisms of craniofacial development to enable the majority of these defects to be detected or prevented pre-natally. Our goal is to develop animal models of craniofacial malformations that will lead to mechanistic insight into the diagnosis and treatment of related human birth defects. We focus on the AP-2 family of transcription factors since these genes provide a link between the genetic and environmental causes of human birth defects affecting the face. In mouse and human, five separate genes encode the various AP-2 proteins. Three of these genes are expressed in the developing mouse face, Tcfap2a, Tcfap2b, and Tcfap2c encoding the proteins AP-2a, AP-2, and AP-2? respectively. The human gene encoding AP-2a is mutated in Branchio-Oculo-Facial Syndrome (BOFS) in which there are orofacial clefting defects. Similarly, mutations in the human gene encoding AP-2 cause Char syndrome, which is characterized by face, heart, and limb defects. Expression of Tcfap2a and Tcfap2c are also responsive to retinoic acid application both in vitro and in vivo and these genes form an important component of the gene regulatory network affected by this powerful teratogen. Single gene knockouts of the three mouse AP-2 genes discussed above all result in death during embryogenesis or soon after birth. Tcfap2b null mice do not display overt facial defects and die due to neurological and kidney problems. Tcfap2c null mice die during gastrulation, and so it has been more difficult to gauge the role of this gene in face formation. Tcfap2a has the most direct link to craniofacial development. The Tcfap2a null embryos display multiple defects including orofacial clefting and hypoplasia of the neural crest derived facial skeleton and this is mainly due to loss of AP-2a function in the surface ectoderm. Although Tcfap2a, Tcfap2b, and Tcfap2c have unique roles in development, recent data show that they also share redundant functions. Indeed, preliminary studies indicate that the facial phenotypes generated by compound knockouts of these genes are far more serious than the individual gene knockouts. Thus, the goal of this application is to determine how these proteins function together to regulate craniofacial formation. In Aim 1, we will generate a mouse model of BOFS, which we hypothesize inhibits AP-2 function via a dominant negative mechanism. In Aims 2 and 3 we will generate and analyze particular knockout combinations of the three AP-2 genes expressed in the face using tissue specific Cre-LoxP technology. The results of these analyses will reveal the combinatorial roles for the AP-2 genes in craniofacial morphogenesis and provide significant insight into the transcriptional control of facial development.